PURPOSE. To elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end–capping protein. METHODS. We investigated F-actin and F-actin–binding protein localization in interdigitations of Tmod1+/+ and Tmod1‒/‒single mature lens fibers. RESULTS. F-actin–rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1‒/‒ mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, β2-spectrin, and a-actinin are localized in large puncta in valleys between paddles; but in Tmod1‒/‒ mature fibers, β2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1‒/‒ mature fibers. CONCLUSIONS. These results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a β2-spectrin–actin network stabilized by Tmod1. α-Actinin–crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled Factin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin– associated proteins required for the formation of paddles between lens fibers.
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience